1. Field of the Invention
This invention provides for a method of quantifying bacteria using a bacterial specific nucleic acid probe which is complementary to a unique, open and highly conserved region of the 16S ribosomal RNA (rRNA) of bacteria. This probe permits the rapid detection of 16S rRNA in a sample and by comparison with known standards, one can estimate the total bacterial count in the sample. The method is accurate, reproducible and conducted at room temperature.
2. Information Disclosure
The use of signal intensity of nucleic acid hybridization assay to estimate total nucleic acid present in a sample is known. Gowans, E. J., Jilbert, A. R. and Burrell, C. J., Detection of Specific DNA and RNA Sequences in Tissues and Cells By In Situ Hybridization (Chapter 5, 1989) in Nucleic Acid Probes, Ed. Symons, R. H., CRC Press, Inc. Boca Raton, Fla.; and Anderson, M. L. M. and Young, B. D.,1987, Quantitative Analysis of Solution Hybridization, (Sec. 10) in Nucleic Acid Hybridization a Practical Approach, Eds. B. D. Hames and S. J. Higgins, IRL Press, Washington D.C. U.S.A.
Universal probes for the detection of bacteria are known. Giovannoni, S. J. et al., 1988, Phylogentic Group-specific Oligodeoxynucleotide Probes for Identification of Single Microbial Cells, J. Bact. 170(2):720-726 and Chuba, P. J. et al., 1988, Synthetic Oligodeoxynucleotide Probes for the Rapid Detection of Bacteria Associated with Human Periodontitis, J. Gen. Microbiol. 134:1931-1938. Oligonucleotides reflecting the UP9A region were described by Woese, C. R., et al., (1975), Conservation of primary structure in 16S ribosomal RNA, Nature 254:83-85 (see Table 1, oligos 47, 49 and 51) and WO 88/03957 (see page 105).
The use of total bacterial count to diagnose periodontal disease is not a presently accepted practice. Socranksky, S. S. et al., The Microbiota of the Gingival Crevice Area of Man-I Total Microscopic and Viable Counts and Counts of Specific Organisms, Arch. Oral. Biol 8:275-280 and Moore, W. E. C., 1987, Microbiology of Periodontal , Disease, J. Periodontal Res. 22:335-341.
Microbial counts were used to determine the effectiveness of tetracycline for prevention of periodontal disease by the Forsyth Center and reported in J. of Dental Res. Annual Session, Mar. 15-19, 1989, Vol. 68, page 197, Abstract Nos. 122-124.